Both cellular and molecular midline guidance cues play a major role in regulating the behavior of growth cones as axonal tracts are established during embryogenesis. The proposed research will address how midline cell types (glial cells) and the midline molecular guidance cue Sema3D influence pathfinding decisions of retinal ganglion cell (RGC) and post optic commissure (POC) axons in the zebrafish forebrain. Based on our preliminary data we hypothesize that a glial bridge exists in the region of the forebrain commissures prior to midline axon crossing and that this bridge forms an important substrate for pioneering growth cones. Preliminary evidence in mutants whose axons fail to cross the forebrain midline (Hedgehog(Hh)/achiasmatic mutants), lends support to this hypothesis, as glial cells are selectively lost in the chiasm/POC and AC midline regions. We propose cell transplantation experiments designed to identify, in an unbiased way, which forebrain midline cells can guide axons across the midline (cellular cues). Gene expression analyses in Hh/achiasmatic mutants has focused our search for molecular cues that guide axons toward the midline on the cell surface molecule Semaphorin3D. We plan to determine whether midline glial cells express Sema3D using immuno- and in situ- co-labeling and using an Olig2-GFP transgenic zebrafish line. Finally, we will use a Heatshock-Sema3D-GFP trangenic zebrafish line to test whether local misexpression of Serm3D in the chiasm is capable of rescuing midline axon crossing in Hh/achiasmatic mutants. This research will lead to a better understanding of events critical to establishing neural connections across the midline in the vertebrate brain.